**2.3. Micropropagation to produce sanitated plants**

Systemic buildup of infections particularly diseases (viral, bacterial and fungal) affect plant vigor and health. Unavailability of an efficient *in vitro* mass multiplication system in sugarcane is a major constraint in the provision of disease free elite germplasm [21]. Sugarcane has long breeding cycle and it requires 10–15 years to complete selection cycle. Fuzz multiplication rate of newly released sugarcane varieties is always slow 1:6–1:8 [22] and diseases accumulate in the seed sets during multiplication. Unavailability of disease free planting material is a major limitation in the improvement of sugarcane crop. Normally, sugarcane reproduces vegetatively but seed propagation is also there under particular climatic conditions [23]. Nodal cuttings are being used for the propagation of commercial sugarcane. In this method of multiplication, meristematic or non-meristematic tissues are used as explant. Sugarcane plants have been regenerated directly from apical and axillary meristems and also from immature leaf tissues [24].

*In vitro* propagation of sugarcane through meristematic tissues responded better as compared with other types of plant tissues. Therefore, significant efforts have been made to explore meristematic tissues for mass multiplication. In the beginning of twenty-first century, some reports highlighted direct regeneration of sugarcane genotypes through thin layer culture of cells from immature leaf or inflorescence [24]. They reported to lessen the time span required for *in vitro* propagation. Significant efforts have been made to establish protocols for direct or indirect sugarcane regeneration. Almost each part of the sugarcane plant has been exploited for callus induction but only inflorescence and immature leaves [25] responded better to morphogenic callus. Callus based regeneration gained significance with the prediction that *in-vitro* induced mutations can play some key role in sugarcane improvement [26]. *In vitro* induced variability is beneficial for the development of new varieties but it becomes undesirable when true-to-type plants are required. Only fewer examples have been quoted to highlight useful variations in callus-derived plants. Meristem culture was successfully used to eradicate chlorotic streak disease, sugarcane mosaic virus [27], white leaf disease and ratoon stunting disease. Combination of meristem culture and heat treatment have proved very effective to eradicate pathogens of Fiji disease [28], SCMV [27] and downy mildew. It is an effective method to eliminate most of the bacterial and fungal diseases and is commonly used to eradicate diseases of unknown etiology as well. Disease free planting material of sugarcane obtained from apices culture is now routinely used for the international exchange of this crop. Researchers have explored that plants regenerated from thin cell layer culture can be used to produce disease free sugarcane plants from the infected ones with *Leifsonia xyli*, SCMV and FDV. *In vitro* culture techniques are used in Brazil and USA to produce healthy planting material for commercial applications. Cryotherapy has also appeared as a proficient method to eliminate phytoplasma from the crop plants and has also been used for long term storage of germplasm or production of disease free plants [29].

#### **2.4. Germplasm conservation**

their approval or field establishment. Various high yielding, early maturing, high sucrose content and smut resistant varieties with good ratooning ability have also been developed

Somaclonal variation have been employed to improve cane-quality, sugar recovery, yield, drought tolerance and disease resistance. To increase the incidence of genetic variation and to get positive modifications in the target plant genome, physical (ion beams, gamma rays) and chemical (sodium azide, sodium nitrite and ethylmethane sulfonate (EMS) mutagens have been tested [15]. Introduction of selection pressure at cellular level has been successful to isolate mutants with desired characters. For fungal pathogen resistance inoculation with fungal pathotoxins or culture filtrates has been very effective. Somaclonal variants of sugarcane were developed against eye spot disease by Larkin and Scowcroft. Mutagenesis has been used by various researchers to isolate embryogenic cells and plants tolerant against red rot [16].

Similarly, for abiotic stress tolerance polyethylene glycol, mannitol and sodium chloride has been used for the selection of plants against drought/salinity tolerance [17]. Various studies were conducted to evaluate the level of variability and transmission of variations into the next generation by vegetative propagation. These studies verified the occurrence of considerable variations in *in-vitro* derived plants. However, extensive field experiments showed that tissue culture derived phenotypic variations were often temporary as most of the variants relapsed to the parental phenotype in the first ratoon crop [18]. Few other studies also supported somaclonal variations but to little extent. Chowdhury and Vasil [19] were not able to recognize any considerable variation in the DNA of plants regenerated from cell suspension, protoplasts and callus cultures. Taylor et al. [20] performed random amplified polymorphic DNA (RAPD) analyses of plants regenerated from embryogenic culture. Only a few polymorphisms were observed. Anyhow, there are different opinions about the somaclonal variations and their transmission to next generation. Most of the researchers are of the view that these variations depend upon the genetic makeup and experimental conditions under which plants are screened and selected.

Systemic buildup of infections particularly diseases (viral, bacterial and fungal) affect plant vigor and health. Unavailability of an efficient *in vitro* mass multiplication system in sugarcane is a major constraint in the provision of disease free elite germplasm [21]. Sugarcane has long breeding cycle and it requires 10–15 years to complete selection cycle. Fuzz multiplication rate of newly released sugarcane varieties is always slow 1:6–1:8 [22] and diseases accumulate in the seed sets during multiplication. Unavailability of disease free planting material is a major limitation in the improvement of sugarcane crop. Normally, sugarcane reproduces vegetatively but seed propagation is also there under particular climatic conditions [23]. Nodal cuttings are being used for the propagation of commercial sugarcane. In this method of multiplication, meristematic or non-meristematic tissues are used as explant. Sugarcane plants have been regenerated directly from apical and axillary meristems and also

through somatic embryogenesis.

**2.3. Micropropagation to produce sanitated plants**

from immature leaf tissues [24].

**2.2. Somaclonal variations**

116 Sugarcane - Technology and Research

Another important application of *in vitro* techniques that attracted researchers is germplasm conservation [30]. *In-vitro* storage of sugarcane germplasm had been established at the Centre de Cooperation Internationale en Recherche Agronomique pour le Development (CIRAD) in France, Sugarcane Breeding Institute in India, and BSES Limited (formerly the Bureau of Sugar Experiment Stations) in Australia. More than 200 hybrid clones of *Saccharum* spp. were preserved at 18°C for 12 months and no phenotypic modifications were observed in the recovered plants. However, with the advancements in tissue culture techniques, *in vitro* preservation became more valuable for the preservation of genetic resources especially of sugarcane [31]. The minimal medium used in *in vitro* preservation has been used successfully during short and medium term preservation, especially for meristems and shoot apices. Decline in explants metabolic activity is usually achieved by changing physical environment or composition of the media used [32]. The commonly used approaches are: lowering of incubation temperature and use of osmotically active compounds such as sorbitol, mannitol and sucrose. Moreover, growth inhibitors like abscisic acid (ABA) is also frequently used. Various factors i.e. vitamins, salts, osmotic stress and others have been explored by different researchers. Survival rate varied in all these experiments but nobody exploited genetic or cytological studies to assess the genetic stability of *in vitro* plants. For diploid species, Simple Sequence Repeat (SSR), Amplified Fragment Length Polymorphism (AFLP) and Inter-Simple Sequence Repeat (ISSR) have been used successfully to assess genetic stability of *in-vitro* plants [33]. But, for plants like sugarcane which has complex polyploid genome, these tools are inappropriate as interpretation of the results become tricky [34]. Using microscopic techniques for sugarcane is also difficult because of small size and large number of chromosomes and also due to the presence of various cytotypes [35]. In this context flow cytometry has got attraction as it ensures estimation of relative amounts of plants nuclear DNA quickly and precisely [36]. Cytometry is able to discriminate between plants derived different culture techniques and has extensively been used, in many economically important species such as *Gossypium hirsutum*, *Vitis vinifera*, *Passiflora* spp., *Elaeis guineensis*, *Musa acuminata* and *Prunus cerasus* [37]. Flow cytometric analysis of shoots was performed after every 6 months of storage. As a consequence, a discrete behavior of tested varieties was observed during storage and on average approximately 80% cultures were able to recover. From these findings it is concluded sugarcane genotypes can be maintained in minimal growth condition for extensive periods but may lead to genetic variations.

between coding genes strongly support the extension of genome in grasses. Transposons and retrotransposons are two categories of transposable elements. In plants, the most abundant retroelements are LTR (long terminal repeats) retrotransposons. Transposase proteins are involved in insertion-deletion mechanisms. Active sites of the transcription control the movement of retrotransposons, which reinsert them into the genome after each propagation cycle to increase copy number. Recent studies show that there exists a gene remodeling mechanism which results in the generation of new genes. As a result of gene remodeling, gene expression is altered by new regulatory networks [41]. The study of transposable elements in wheat and barley [42] provided close relationship of transposable elements with genome structure. The transposable elements in sugarcane can be activated and evaluated by functional transcriptomic approaches. The major limitation in sugarcane genetic improvement is its genome size. To sequence the genome of an organism Bacterial Artificial Chromosomes (BAC) are used. BAC (Bacterial Artificial Chromosomes) library was constructed with Hin*d*III partial digestion for sugarcane cultivar 'R570' having more than 100,000 clones with 130 mega base pairs (Mb) insert size [43]. For map-based cloning of sugarcane, BAC resources will be highly esteemed and physical map of sorghum (http://www.genome.clemson.edu/tools/contig\_viewer/index.

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Transcriptomic approaches have emerged as an effective tool for functional characterization of unknown genes. In combination with proteomics and metabolomics, these approaches are very useful for the development of improved sugarcane clones. It reduces the complexity of data and targets. Only active genes in the cell or tissues are considered at the time of sampling. By employing transcriptomic approaches, one can easily compare similar type of tissues at different developmental stages in different organisms growing in different conditions [44].

Due to large size and complexity of genome, the whole genome sequence of sugarcane was not available. The genome size of its modern cultivar is considered to be more than 10 GB. From 12th July, 2017, NCBI database has 83,138 GSSs (genome survey sequences), 285,216 ESTs (expressed sequence tags) and 13,382 nucleotide sequences including 491 sequences of mRNA under the search of "*Saccharum*". There are three main groups of ESTs including a large group resulting from a modern variety of sugarcane and two small groups from *Saccharum officinarum* and *Saccharum arundinaceum* (**Table 1**). Majority of ESTs belong to six cultivars from different countries including Australia (Q117), USA (CP72-2086), India (CoS 767, Co 1148) and Brazil (SP80-3280, SP70-1143). Most of the ESTs are from mixed tissue samples of Brazilian varieties i.e. P57150-4 x PB5211 or SP83-5077, RB80-5028; SP80-87432, RB855205, CB47-89, RB845298,

Many projects have been executed for sequencing sugarcane ESTs (expressed sequence tags) in Brazil (http://sucest.lad.ic.unicamp.br/en), South Africa and Australia [46]. Until now more than 0.3 million (300,000) ESTs have been generated. A database holding 0.238 million (238,000) ESTs (constructed from diverse organs and tissues) from 37 libraries was erected by

SP803280 x SP81-5441, SP80-185, SP80-3280 and SP87-396 [45].

html) will be used as complementary tool.

**3.2. Transcriptomics**

**3.3. ESTs**
